Chemokine profiling in children and adults with symptomatic and asymptomatic respiratory viral infections.

Molecular diagnosis; Viral infection; Chemokines; Disease prognosis; CXCL10; CXCL11; CCL3; CCL4; CCL5; Random forest.

Surveillance-embedded genomic outbreak resolution of methicillin-susceptible Staphylococcus aureus in a neonatal intensive care unit.

We observed an increase in methicillin-susceptible Staphylococcus aureus (MSSA) infections at a Dutch neonatal intensive care unit. Weekly neonatal MSSA carriage surveillance and cross-sectional screenings of health care workers (HCWs) were available for outbreak tracing. Traditional clustering of MSSA isolates by spa typing and Multiple-Locus Variable number tandem repeat Analysis (MLVA) suggested that nosocomial transmission had contributed to the infections. We investigated whether whole-genome sequencing (WGS) of MSSA surveillance would provide additional evidence for transmission. MSSA isolates from neonatal infections, carriage surveillance, and HCWs were subjected to WGS and bioinformatic analysis for identification and localization of high-quality single nucleotide polymorphisms, and in-depth analysis of subsets of isolates. By measuring the genetic diversity in background surveillance, we defined transmission-level relatedness and identified isolates that had been unjustly assigned to clusters based on MLVA, while spa typing was concordant but of insufficient resolution. Detailing particular subsets of isolates provided evidence that HCWs were involved in multiple outbreaks, yet it alleviated concerns about one particular HCW. The improved resolution and accuracy of genomic outbreak analyses substantially altered the view on outbreaks, along with apposite measures. Therefore, inclusion of the circulating background population has the potential to overcome current issues in genomic outbreak inference.

Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck.

Antifungal susceptibility testing is an essential tool for guiding therapy, although EUCAST and CLSI reference methods are often available only in specialized centers. We studied the performance of an agar-based screening method for the detection of azole resistance in Aspergillus fumigatus cultures. The VIPcheck consists of four wells containing voriconazole, itraconazole, posaconazole, or a growth control. Ninety-six A. fumigatus isolates were used. Thirty-three isolates harbored a known resistance mechanism: TR34/L98H (11 isolates), TR46/Y121F/T289A (6 isolates), TR53 (2 isolates), and 14 isolates with other cyp51A gene point mutations. Eighteen resistant isolates had no cyp51A-mediated azole resistance. Forty-five isolates had a wild-type (WT) azole phenotype. Four technicians and two inexperienced interns, blinded to the genotype/phenotype, read the plates visually after 24 h and 48 h and documented minimal growth, uninhibited growth, and no growth. The performance was compared to the EUCAST method. After 24 h of incubation, the mean sensitivity and specificity were 0.54 and 1.00, respectively, with uninhibited growth as the threshold. After 48 h of incubation, the performance mean sensitivity and specificity were 0.98 and 0.93, respectively, with minimal growth. The performance was not affected by observer experience in mycology. The interclass correlation coefficient was 0.87 after 24 h and 0.85 after 48 h. VIPcheck enabled the selection of azole-resistant A. fumigatus colonies, with a mean sensitivity and specificity of 0.98 and 0.93, respectively. Uninhibited growth on any azole-containing well after 24 h and minimal growth after 48 h were indicative of resistance. These results indicate that the VIPcheck is an easy-to-use tool for azole resistance screening and the selection of colonies that require MIC testing.

[Skeletal muscle ischemia-reperfusion and ischemic conditioning pathophysiology-clinical applications for the vascular surgeon]. / Physiopathologie de l'ischémie-reperfusion et du conditionnement ischémique du muscle squelettique ­ applications cliniques pour le chirurgien vasculaire.

Ischemia-reperfusion, which is characterized by deficient oxygen supply and subsequent restoration of blood flow, can cause irreversible damage to tissue. The vascular surgeon is daily faced with ischemia-reperfusion situations. Indeed, arterial clamping induces ischemia, followed by reperfusion when declamping. Mechanisms underlying ischemia-reperfusion injury are complex and multifactorial. Increases in cellular calcium and reactive oxygen species, initiated during ischemia and then amplified upon reperfusion are thought to be the main mediators of reperfusion injury. Mitochondrial dysfunction also plays an important role. Extensive research has focused on increasing skeletal muscle tolerance to ischemia-reperfusion injury, especially through the use of ischemic conditioning strategies. The purpose of this review is to focus on the cellular responses associated with ischemia-reperfusion, as well as to discuss the effects of ischemic conditioning strategies. This would help the vascular surgeon in daily practice, in order to try to improve surgical outcome in the setting of ischemia-reperfusion.

Cardiac mitochondrial oxidative capacity is partly preserved after cryopreservation with dimethyl sulfoxide.

BACKGROUND: Cardiac muscle cryopreservation is a challenge for both diagnostic procedure requiring viable tissues and therapeutic advance in regenerative medicine. Mitochondria are targets of both direct and indirect damages, secondary to congelation per se and/or to cryoprotectant's toxic effects, which participate to diminution of viability and/or functioning of cells after freezing. At the cardiac muscle level, only one study had investigated mitochondrial respiration after cryopreservation. OBJECTIVE: To determine the effect of cryopreservation on mitochondrial respiration of cardiac muscle. MATERIALS AND TMETHODS: We recorded mitochondrial respiration through complexes I, II, III and IV along with mitochondrial coupling in fresh and cryopreserved rat left ventricles samples and assessed difference of the means, correlation and agreement between the measures in all samples. RESULTS: Mitochondrial respiration was partly maintained up to 70% in cryopreserved samples whatever the substrate. A significant correlation was observed between fresh and cryopreserved samples (r = 0.71, p < 0.0001). However, mitochondrial coupling significantly decreased after cryopreservation (- 1.44 ± 0.15; p < 0.005) suggesting that mitochondrial intactness was not totally preserved by cryopreservation. Further, the fluctuations around the mean difference were wide (-14.06, +5.08 µmol/min/g), increasing with respiration rates (p < 0.0001). CONCLUSION: Thus, fresh samples extemporaneous analysis should be preferred when available despite the fact that cryopreservation using DMSO partly protect cardiac mitochondrial respiration and coupling. These data support the interest to further refine cryopreservation methods.

A new murine model of sustainable and durable chronic critical limb ischemia fairly mimicking human pathology.

OBJECTIVE: To establish a chronic mouse model of critical limb ischemia (CLI) with in vivo and ex vivo validation, closely mimicking human pathology. METHODS: Swiss mice (n = 28) were submitted to sequential unilateral femoral (day 0) and iliac (day 4) ligatures. Ischemia was confirmed by clinical scores (tissue and functional damages) and methoxyisobutylisonitrile (MIBI) scintigraphies at days 0, 4, 6, 10, 20, and 30. At days 10, 20, and 30, muscle mitochondrial respiration, calcium retention capacity (CRC), and production of reactive oxygen species (ROS) were investigated, together with transcripts of mitochondrial biogenesis and antioxidant enzymes. Histological analysis was also performed. RESULTS: Clinical and functional damage confirmed CLI. MIBI scintigraphies showed hypoperfusion of the ischemic limb, which remained stable until day 30. Mitochondrial respiration was impaired in ischemic muscles compared with controls (Vmax = 7.93 ± 0.99 vs. 10.09 ± 2.87 mmol/L O2/minute/mg dry weight [dw]; p = .01), together with impaired CRC (7.4 ± 1.6 mmol/L minute/mg dw vs. 11.9 ± 0.9 mmol/L minute/mg dw; p < .001) and biogenesis (41% decrease in peroxisome proliferator-activated receptor gamma coactivator [PGC]-1α, 49% decrease in PGC-1ß, and 41% decrease in nuclear respiratory factor-1). Ischemic muscles also demonstrated increased production of ROS under electron paramagnetic resonance (0.084 ± 0.029 vs. 0.051 ± 0.031 mmol/L minute/mg dw; p = .03) and with dihydroethidium staining (3622 ± 604 arbitrary units of fluorescence vs. 1224 ± 324; p < .01), decreased antioxidant enzymes (32% decrease in superoxide dismutase [SOD]1, 41% decrease in SOD2, and 49% decrease in catalase), and myopathic features (wider range in fiber size, rounded shape, centrally located nuclei, and smaller cross-sectional areas). All defects were stable over time. CONCLUSION: Sequential femoral and iliac ligatures closely mimic human functional, clinical, scintigraphic, and skeletal muscle mitochondrial characteristics, and could prove useful for testing therapeutic approaches.

Compartmentalization of Inflammatory Response Following Gut Ischemia Reperfusion.

OBJECTIVE/BACKGROUND: Gut ischemia reperfusion (IR) is thought to trigger systemic inflammation, multiple organ failure, and death. The aim of this study was to investigate inflammatory responses in blood and in two target organs after gut IR. METHODS: This was a controlled animal study. Adult male Wistar rats were randomized into two groups of eight rats: control group and gut IR group (60 minutes of superior mesenteric artery occlusion followed by 60 minutes of reperfusion). Lactate and four cytokines (tumor necrosis factor-a, interleukin [IL]-1b, IL-6, and IL-10) were measured in mesenteric and systemic blood. The relative gene expression of these cytokines was determined by real time polymerase chain reaction in the gut, liver, and lung. RESULTS: Gut IR significantly increased lactate levels in mesenteric (0.9 ± 0.4 vs. 3.7 ± 1.8 mmol/L; p < .001) and in systemic blood (1.3 ± 0.2 vs. 4.0 ± 0.3 mmol/L; p < .001). Gut IR also increased the levels of four cytokines in mesenteric and systemic blood. IL-6 and IL-10 were the main circulating cytokines; there were no significant differences between mesenteric and systemic cytokine levels. IL-10 was upregulated mainly in the lung,suggesting that this organ could play a major role during gut reperfusion. CONCLUSION: The predominance of IL-10 over other cytokines in plasma and the dissimilar organ responses,especially of the lung, might be a basis for the design of therapies, for example lung protective ventilation strategies, to limit the deleterious effects of the inflammatory cascade. A multi-organ protective approach might involve gut directed therapies, protective ventilation, hemodynamic optimization, and hydric balance.

In vitro antifungal susceptibility of Trichophyton violaceum isolated from tinea capitis patients.

OBJECTIVES: Trichophyton violaceum is an anthropophilic dermatophyte that is endemic to parts of Africa and Asia and is sporadic in Europe. T. violaceum mainly causes tinea capitis in both children and adolescents. Although the infections caused by T. violaceum are of considerable medical importance, its antifungal susceptibility profile remains poorly examined. METHODS: In this study, we tested the in vitro antifungal susceptibility of a set of clinical T. violaceum isolates obtained from tinea capitis patients, using the CLSI broth microdilution method. We tested eight antifungals and used isolates collected from Western China (21), Eastern China (12), the Middle East (1), Europe (20), South Africa (7) and Canada (1). RESULTS: The geometric means of the MICs of the antifungals for all isolates were as follows (in increasing order): posaconazole, 0.021 mg/L; terbinafine, 0.023 mg/L; voriconazole, 0.062 mg/L; amphotericin B, 0.20 mg/L; itraconazole, 0.34 mg/L; caspofungin, 0.56 mg/L; fluconazole, 4.23 mg/L; and flucytosine, 8.46 mg/L. No statistically significant differences in the susceptibility profiles of T. violaceum were detected within the geographical regions tested. CONCLUSIONS: Posaconazole, terbinafine and voriconazole were shown to be the most potent antifungal agents against T. violaceum isolates obtained from tinea capitis patients worldwide. These results might help clinicians in developing appropriate therapies that have a high probability of successfully treating tinea capitis due to T. violaceum.

Remote effects of lower limb ischemia-reperfusion: impaired lung, unchanged liver, and stimulated kidney oxidative capacities.

Remote organ impairments are frequent and increase patient morbidity and mortality after lower limb ischemia-reperfusion (IR). We challenged the hypothesis that lower limb IR might also impair lung, renal, and liver mitochondrial respiration. Two-hour tourniquet-induced ischemia was performed on both hindlimbs, followed by a two-hour reperfusion period in C57BL6 mice. Lungs, liver and kidneys maximal mitochondrial respiration (V(max)), complexes II, III, and IV activity (V(succ)), and complex IV activity (V(TMPD)) were analyzed on isolated mitochondria. Lower limb IR decreased significantly lung V(max) (29.4 ± 3.3 versus 24 ± 3.7 µmol O2/min/g dry weight, resp.; P = 0.042) and tended to reduce V(succ) and V(TMPD). IR did not modify liver but increased kidneys mitochondrial respiration (79.5 ± 19.9 versus 108.6 ± 21.4, P = 0.035, and 126 ± 13.4 versus 142.4 ± 10.4 µmol O2/min/g dry weight for V(max) and V(succ), resp.). Kidneys mitochondrial coupling was increased after IR (6.5 ± 1.3 versus 8.8 ± 1.1, P = 0.008). There were no histological changes in liver and kidneys. Thus, lung mitochondrial dysfunction appears as a new early marker of hindlimb IR injuries in mice. Further studies will be useful to determine whether enhanced kidneys mitochondrial function allows postponing kidney impairment in lower limb IR setting.

Mitochondrial uncoupling reduces exercise capacity despite several skeletal muscle metabolic adaptations.

The effects of mitochondrial uncoupling on skeletal muscle mitochondrial adaptation and maximal exercise capacity are unknown. In this study, rats were divided into a control group (CTL, n = 8) and a group treated with 2,4-dinitrophenol, a mitochondrial uncoupler, for 28 days (DNP, 30 mg·kg(-1)·day(-1) in drinking water, n = 8). The DNP group had a significantly lower body mass (P < 0.05) and a higher resting oxygen uptake (Vo2, P < 0.005). The incremental treadmill test showed that maximal running speed and running economy (P < 0.01) were impaired but that maximal Vo2 (Vo2max) was higher in the DNP-treated rats (P < 0.05). In skinned gastrocnemius fibers, basal respiration (V0) was higher (P < 0.01) in the DNP-treated animals, whereas the acceptor control ratio (ACR, Vmax/V0) was significantly lower (P < 0.05), indicating a reduction in OXPHOS efficiency. In skeletal muscle, DNP activated the mitochondrial biogenesis pathway, as indicated by changes in the mRNA expression of PGC1-α and -ß, NRF-1 and -2, and TFAM, and increased the mRNA expression of cytochrome oxidase 1 (P < 0.01). The expression of two mitochondrial proteins (prohibitin and Ndufs 3) was higher after DNP treatment. Mitochondrial fission 1 protein (Fis-1) was increased in the DNP group (P < 0.01), but mitofusin-1 and -2 were unchanged. Histochemical staining for NADH dehydrogenase and succinate dehydrogenase activity in the gastrocnemius muscle revealed an increase in the proportion of oxidative fibers after DNP treatment. Our study shows that mitochondrial uncoupling induces several skeletal muscle adaptations, highlighting the role of mitochondrial coupling as a critical factor for maximal exercise capacities. These results emphasize the importance of investigating the qualitative aspects of mitochondrial function in addition to the amount of mitochondria.

Aspergillosis due to voriconazole highly resistant Aspergillus fumigatus and recovery of genetically related resistant isolates from domiciles.

BACKGROUND: Azole resistance is an emerging problem in Aspergillus fumigatus and complicates the management of patients with Aspergillus-related diseases. Selection of azole resistance may occur through exposure to azole fungicides in the environment. In the Netherlands a surveillance network was used to investigate the epidemiology of resistance selection in A. fumigatus. METHODS: Clinical A. fumigatus isolates were screened for azole resistance in 8 university hospitals using azole agar dilution plates. Patient information was collected using an online questionnaire and azole-resistant A. fumigatus isolates were analyzed using gene sequencing, susceptibility testing, and genotyping. Air sampling was performed to investigate the presence of resistant isolates in hospitals and domiciles. RESULTS: Between December 2009 and January 2011, 1315 A. fumigatus isolates from 921 patients were screened. A new cyp51A-mediated resistance mechanism (TR46/Y121F/T289A) was observed in 21 azole-resistant isolates from 15 patients in 6 hospitals. TR46/Y121F/T289A isolates were highly resistant to voriconazole (minimum inhibitory concentration ≥16 mg/L). Eight patients presented with invasive aspergillosis due to TR46/Y121F/T289A, and treatment failed in all 5 patients receiving primary therapy with voriconazole. TR46/Y121F/T289A Aspergillus fumigatus was recovered from 6 of 10 sampled environmental sites. CONCLUSIONS: We describe the emergence and geographical migration of a voriconazole highly resistant A. fumigatus that was associated with voriconazole treatment failure in patients with invasive aspergillosis. Recovery of TR46/Y121F/T289A from the environment suggests an environmental route of resistance selection. Exposure of A. fumigatus to azole fungicides may facilitate the emergence of new resistance mechanisms over time, thereby compromising the use of azoles in the management of Aspergillus-related diseases.

Methylene blue protects liver oxidative capacity after gut ischaemia-reperfusion in the rat.

OBJECTIVES: Mesenteric ischaemia/reperfusion (IR) may lead to liver mitochondrial dysfunction and multiple organ failure. We determined whether gut IR induces early impairment of liver mitochondrial oxidative activity and whether methylene blue (MB) might afford protection. DESIGN: Controlled animal study. MATERIALS AND METHODS: Rats were randomised into three groups: controls (n = 18), gut IR group (mesenteric ischaemia (60 min)/reperfusion (60 min)) (n = 18) and gut IR + MB group (15 mg kg(-1) MB intra-peritoneally) (n = 16). Study parameters were: serum liver function markers, blood lactate, standard histology and DNA fragmentation (apoptosis) on intestinal and liver tissue, maximal oxidative capacity of liver mitochondria (state 3) and activity of complexes II, III and IV of the respiratory chain measured using a Clark oxygen electrode. RESULTS: Gut IR increased lactate deshydrogenase (+982%), aspartate and alanine aminotransferases (+43% and +74%, respectively) and lactate levels (+271%). It induced segmental loss of intestinal villi and cryptic apoptosis. It reduced liver state 3 respiration by 30% from 50.1 ± 3 to 35.2 ± 3.5 µM O(2) min(-1) g(-1) (P < 0.01) and the activity of complexes II, III and IV of the mitochondrial respiratory chain. Early impairment of liver mitochondrial respiration was related to blood lactate levels (r(2) = 0.45). MB restored liver mitochondrial function. CONCLUSIONS: MB protected against gut IR-induced liver mitochondria dysfunction.

Local but not systemic capillary lactate is a reperfusion biomarker in experimental acute limb ischaemia.

INTRODUCTION: Systemic capillary lactate, an end product of cellular anaerobic metabolism, has not established credibility in monitoring limb reperfusion. We assessed, in mice, whether local capillary lactate, arising from the reperfused limb, might be a relevant biomarker of reperfusion. REPORT: Systemic and local capillary lactate were sampled in the non-ischaemic and in the ischaemic limb. Only local lactate concentrations significantly increased after 2 h of ischaemia and decreased after reperfusion. DISCUSSION: Local, but not systemic, capillary lactate appeared as a potential reperfusion biomarker in this experimental acute limb ischaemia model.

Effect of postconditioning on mitochondrial dysfunction in experimental aortic cross-clamping.

BACKGROUND: Cross-clamping of the aorta during abdominal aortic aneurysm surgery induces muscle ischaemia with resultant morbidity. This study tested whether ischaemic postconditioning would decrease mitochondrial dysfunction in skeletal muscle by reducing oxidative stress. METHODS: Three groups (9 rats each) underwent surgery, including a control group without ischaemia and an ischaemia-reperfusion group that had 3 h ischaemia induced by aortic clamping and collateral vessel ligation, followed by 2 h of reperfusion. The third group had ischaemia for 3 h then underwent postconditioning comprising three short intervals of ischaemia-reperfusion at the onset of reperfusion. Activity of complexes I, II, III and IV of the mitochondrial respiratory chain was monitored in gastrocnemius muscle, along with oxidative stress measured by dihydroethidium (DHE) staining and antioxidant defence determined by measurement of glutathione levels. RESULTS: Ischaemia-reperfusion alone caused a significant reduction in maximal oxidative capacity (-31.8 per cent; P = 0.002), activity of complexes II, III and IV (-34.5 per cent; P = 0.007) and complex IV activity (-30.6 per cent; P = 0.039). It also increased reactive oxygen species (DHE staining increased to 223.1 per cent of control value; P = 0.027) and reduced antioxidant defence (glutathione level -28.6 per cent; P = 0.039). Postconditioning counteracted these deleterious effects by increasing mitochondrial complex I, II, III and IV activities, restoring muscle DHE staining and preserving glutathione content. CONCLUSION: Ischaemic postconditioning protects skeletal muscle mitochondria against ischaemia-reperfusion injury by reducing oxidative stress and preserving antioxidant defence in an experimental model. Mitochondrial protection to reduce reperfusion injury in clinical vascular surgery may be warranted.

Heart failure: a model of cardiac and skeletal muscle energetic failure.

Chronic heart failure (CHF), the new epidemic in cardiology, is characterized by energetic failure of both cardiac and skeletal muscles. The failing heart wastes energy due to anatomical changes that include cavity enlargement, altered geometry, tachycardia, mitral insufficiency and abnormal loading, while skeletal muscle undergoes atrophy. Cardiac and skeletal muscles also have altered high-energy phosphate production and handling in CHF. Nevertheless, there are differences in the phenotype of myocardial and skeletal muscle myopathy in CHF: cardiomyocytes have a lower mitochondrial oxidative capacity, abnormal substrate utilisation and intracellular signalling but a maintained oxidative profile; in skeletal muscle, by contrast, mitochondrial failure is less clear, and there is altered microvascular reactivity, fibre type shifts and abnormalities in the enzymatic systems involved in energy distribution. Underlying these phenotypic abnormalities are changes in gene regulation in both cardiac and skeletal muscle cells. Here, we review the latest advances in cardiac and skeletal muscle energetic research and argue that energetic failure could be taken as a unifying mechanism leading to contractile failure, ultimately resulting in skeletal muscle energetic failure, exertional fatigue and death.

Gene expression in skeletal muscle of coronary artery disease patients after concentric and eccentric endurance training.

Low-intensity concentric (CET) and eccentric (EET) endurance-type training induce specific structural adaptations in skeletal muscle. We evaluated to which extent steady-state adaptations in transcript levels are involved in the compensatory alterations of muscle mitochondria and myofibrils with CET versus EET at a matched metabolic exercise intensity of medicated, stable coronary patients (CAD). Biopsies were obtained from vastus lateralis muscle before and after 8 weeks of CET (n=6) or EET (n=6). Transcript levels for factors involved in mitochondrial biogenesis (PGC-1alpha, Tfam), mitochondrial function (COX-1, COX-4), control of contractile phenotype (MyHC I, IIa, IIx) as well as mechanical stress marker (IGF-I) were quantified using an reverse-transcriptase polymerase chain reaction approach. After 8 weeks of EET, a reduction of the COX-4 mRNA level by 41% and a tendency for a drop in Tfam transcript concentration (-33%, P=0.06) was noted. This down-regulation corresponded to a drop in total mitochondrial volume density. MyHC-IIa transcript levels were specifically decreased after EET, and MyHC-I mRNA showed a trend towards a reduction (P=0.08). Total fiber cross-sectional area was not altered. After CET and EET, the IGF-I mRNA level was significantly increased. The PGC-1alpha significantly correlated with Tfam, and both PGC-1alpha and Tfam significantly correlated with COX-1 and COX-4 mRNAs. Post-hoc analysis identified significant interactions between the concurrent medication and muscular transcript levels as well as fiber size. Our findings support the concept that specific transcriptional adaptations mediate the divergent mitochondrial response of muscle cells to endurance training under different load condition and indicate a mismatch of processes related to muscle hypertrophy in medicated CAD patients.

Acute myocardial ischaemia induces specific alterations of ventricular mitochondrial function in experimental pigs.

AIMS: As cardiac metabolic flexibility is crucial, this study examined whether acute ischaemia can induce specific qualitative alterations of the mitochondrial metabolic pathways as well as energy transfer systems. METHODS: Left descending coronary artery ligation was performed after sternotomy in eight pigs and the heart was excised after 45 min of ischaemia. Maximal O2 uptake (V(max), micromol O2 min(-1) g(-1) dry weight) of saponin-skinned myofibres were measured from ischaemic and non-ischaemic area of ventricular myocardium. RESULTS: V(max) decreased by approximately 20% in ischaemic myocardium with both glutamate-malate (18.1 +/- 1.3 vs. 22.1 +/- 1.7 in control, P < 0.05) and pyruvate substrates (19.3 +/- 1.0 vs. 23.3 +/- 2.0 in control, P < 0.05) whereas no difference was observed with palmitoyl carnitine (15.6 +/- 1.8 vs. 16.6 +/- 0.9 in control). The K(m) of mitochondrial respiration for ADP decreased in ischaemic heart by 24% (679 +/- 79 vs. 899 +/- 84 microm of ADP in control, P < 0.05). Moreover, the mitochondrial creatine kinase efficacy (K(m) without creatine/K(m) with creatine), representative of the coupling of oxidative phosphorylation process with the mitochondrial creatine kinase, was reduced in ischaemic heart (11.6 +/- 2.5 in ischaemic vs. 18.0 +/- 2.2 in control, P < 0.05). CONCLUSIONS: These findings argue for specific mitochondrial impairments at the level of pyruvate oxidation and creatine kinase channelling system after an acute period of in vivo ischaemia, whereas the lipid mitochondrial oxidation pathway seems to be preserved. Such a loss of metabolic flexibility following acute ischaemia could become an early feature of metabolic dysregulation of the heart.

Mitochondrial tissue specificity of substrates utilization in rat cardiac and skeletal muscles.

As energetic metabolism is crucial for muscles, they develop different adaptations to respond to fluctuating demand among muscle types. Whereas quantitative characteristics are known, no study described simultaneously quantitative and qualitative differences among muscle types in terms of substrates utilization patterns. This study thus defined the pattern of substrates preferential utilization by mitochondria from glycolytic gastrocnemius (GAS) and oxidative soleus (SOL) skeletal muscles and from heart left ventrical (LV) in rats. We measured in situ, ADP (2 mM)-stimulated, mitochondrial respiration rates from skinned fibers in presence of increasing concentrations of pyruvate (Pyr) + malate (Mal), palmitoyl-carnitine (Palm-C) + Mal, glutamate (Glut) + Mal, glycerol-3-phosphate (G3-P), lactate (Lact) + Mal. Because the fibers oxygen uptake (Vs) followed Michaelis-Menten kinetics in function of substrates level we determined the Vs and Km, representing maximal oxidative capacity and the mitochondrial sensibility for each substrate, respectively. Vs were in the order GAS < SOL < LV for Pyr, Glu, and Palm-C substrates, whereas in the order SOL = LV < GAS with G3-P. Moreover, the relative capacity to oxidize Palm-C is extremely higher in LV than in SOL. Vs was not stimulated by the Lact substrate. The Km was equal for Pyr among muscles, but much lower for G3-P in GAS and lower for Palm-C in LV. These results demonstrate qualitative mitochondrial tissue specificity for metabolic pathways. Mitochondria of glycolytic muscle fibers are well adapted to play a central role for maintaining a satisfactory cytosolic redox state in these fibers, whereas mitochondria of LV developed important capacities to use fatty acids.